Cytidine Base Editors Research Articles

Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts.-Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

CRISPR base editors make mistakes 

Papers from two independent groups suggest that a form of CRISPR gene editing, called base editing, causes a high number of unpredictable mutations in mouse embryos and rice. It’s not the first red flag for CRISPR. Other groups have raised concerns about off-target mutations caused when the traditional CRISPR-Cas9 form of gene editing cuts DNA at a location that it wasn’t supposed to. The results of the new studies are surprising, however, because scientists have lauded base editors as one of the most precise forms of gene editing yet. Base editors change one nucleotide, or letter, of DNA into another without cutting the DNA. They come in two flavors: cytidine base editors, which convert a cytosine (C) nucleotide to a thymine (T), and adenine base editors, which convert an adenosine (A) to a guanosine (G). Both variants were created in David Liu’s lab at Broad Institute of MIT and Harvard.

Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor.

Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.

Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.

Base editors enable targeted single-nucleotide conversion in genomic DNA. Here we show that expression levels are a bottleneck in base editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.

Highly efficient RNA-guided base editing in rabbit

Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53–88% and 44–100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.

Lectures on heaven: an excursion into the playground of the theologians.

<h3>Abstract</h3> Rapid assessment of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based tools and validation of cloned CRISPR-plasmid vectors are critical aspects for the successful application of genome editing (GE) in different organisms, including plants. Also, the selection of active guide RNAs (gRNAs) is a determining factor to achieve efficient GE at the targeted locus. Incidentally, plasmid vectors capable of ectopic expression of Cas enzyme and sgRNAs in bacteria will facilitate the quick screening and reliability of cloned plasmids and the functionality of designed gRNAs. We report a platform for <i>in vivo</i> rapid investigation of CRISPR components in <i>Escherichia coli</i> (IRI-CCE), which is compatible with plant-transforming binary vectors comprising plant promoters/terminators. Besides, IRI-CCE analyses reveal distinct features of cytidine (PmCDA1, evoCDA1, APOBEC3A) and adenine (ABE8e) base editors. Finally, we show the IRI-CCE platform as a reliable and rapid method for screening functional gRNAs to achieve successful GE outcomes. Base editor-based CRISPR components expressed by promoters of different strengths led to the establishment of IRI-CCE platform for three major applications: optimization of novel CRISPR tools, validation of cloned CRISPR plasmids, and to know gRNA functionality.